Anti-silkworm nuclear polyhedrosis virus drug and its application

Abstract

The present invention discloses an anti-Bombyx mori nuclear polyhedrosis virus (Bombyx mori) drug and its application. Experimental results show that artesunate and dihydroartemisinin have anti-Bombyx mori nuclear polyhedrosis virus (Bombyx mori) virus effects and can limit the intracellular proliferation of the virus by inhibiting Bombyx mori nuclear polyhedrosis virus-induced cellular autophagy. These drugs can be used as new anti-Bombyx mori nuclear polyhedrosis virus drugs for silkworms, providing a drug for preventing and treating Bombyx mori blood-type pus disease.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-bombyx mori nuclear polyhedrosis virus drug and an application thereof. Background Art

[0002] Bombyx mori NPV (BmNPV) is the pathogen of blood-borne pyogenic disease in the silkworm, which is characterized by rapid onset and difficulty in control. Currently, there is no effective drug for its prevention and treatment. Therefore, there is an urgent need to find new and effective anti-BmNPV drugs. Summary of the Invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide an anti-Bombyx mori nuclear polyhedrosis virus drug and its application.

[0004] The first object of the present invention is to provide the use of artesunate in preparing a drug for resisting blood-type pus disease of silkworm.

[0005] Preferably, the anti-silkworm blood-type pus disease drug is an anti-silkworm nuclear polyhedrosis virus drug.

[0006] Preferably, the anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cellular autophagy.

[0007] The second object of the present invention is to provide the use of dihydroartemisinin in the preparation of a drug for resisting blood-type pus disease in silkworms.

[0008] Preferably, the anti-silkworm blood-type pus disease drug is an anti-silkworm nuclear polyhedrosis virus drug.

[0009] Preferably, the anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cellular autophagy.

[0010] The present invention has the following beneficial effects:

[0011] Experimental results of the present invention show that artesunate and dihydroartemisinin can inhibit the proliferation of Bombyx mori nuclear polyhedrosis virus (Bombyx mori) and inhibit autophagy induced by Bombyx mori nuclear polyhedrosis virus infection in silkworm ovarian cells. Artesunate and dihydroartemisinin have anti-Bombyx mori nuclear polyhedrosis virus (Bombyx mori) and can limit the intracellular proliferation of the virus by inhibiting Bombyx mori nuclear polyhedrosis virus-induced cellular autophagy. They can be used as new drugs for silkworms to treat Bombyx mori nuclear polyhedrosis virus, providing a drug for the prevention and treatment of blood-type pus disease in silkworms. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1The CCK8 assay detected the effects of different concentrations of drugs on the growth of BmN cells. Note: The data in the figure are mean ± SD, n = 4; *p < 0.05, significant difference; **p < 0.01, ***p < 0.005, extremely significant difference.

[0013] Figure 2 The effects of different drugs on the proliferation of EGFP-BmNPV in BmN cells.

[0014] Figure 3 The inhibitory effect of drug 1 on autophagy in BmN cells was detected by WB.

[0015] Figure 4 The effect of drug 1 on BmNPV proliferation in BmN cells; Note: a: The effect of 10 μmol / L drug 1 treatment on BmNPV proliferation was observed under a fluorescence microscope; b: Western Blot detection of the effect of drug 1 on cell autophagy and viral EGFP-BmNPV proliferation; c: Virus titer at different time points after drug 1 treatment; d: Grayscale scanning data analysis of the Western Blot detection results corresponding to b.

[0016] Figure 5 The effect of drug 3 on BmNPV proliferation in BmN cells was observed under a fluorescence microscope. The effect of drug 3 on BmNPV proliferation was observed under a fluorescence microscope. The effect of drug 3 on cell autophagy and viral EGFP-BmNPV proliferation was detected by Western Blot. The grayscale scanning data analysis of the Western Blot test results in b was obtained. DETAILED DESCRIPTION

[0017] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0018] In the following examples, the experimental drugs were Drug 1 (artesunate), Drug 2 (artemisinin), Drug 3 (dihydroartemisinin), and Drug 4 (artemether). The results showed that Drugs 1 and 3 inhibited autophagy in BmN cells and the proliferation of BmNPV, while Drugs 2 and 4 had no significant inhibitory effect on BmNPV. These results demonstrate that both Drugs 1 and 3 have anti-BmNPV effects in Bombyx mori BmN cells, providing a theoretical basis for the subsequent screening of new anti-BmNPV drugs.

[0019] Example 1

[0020] 1. Experimental Methods

[0021] 1. CCK8 assay to test drug toxicity to BmN cells (silkworm ovary cells)

[0022] The toxicity of drugs to BmN cells was detected using the Cell Counting Kit-8 (CCK-8) kit. The specific steps are as follows:

[0023] (1) Add 100 μL (about 2000) BmN cells to a 96-well cell culture plate, add 3 μL of drugs at different concentrations, treat eight wells with each drug, and set up a DMSO control. Culture continuously at 28°C for 48 h.

[0024] (2) Add 10 μL of CCK-8 solution to each well in a dark environment.

[0025] (3) Incubate at 28°C for 3.5 hours.

[0026] (4) Measure absorbance at a wavelength of 450 nm.

[0027] 2. Preparation of EGFP-BmNPV Recombinant Virus

[0028] A Bac-to-Bac system was used to construct a recombinant BmNPV virus with green fluorescence. The recombinant transfer vector pFastBacHTb-EGFP was transformed into Escherichia coli DH10Bac competent cells containing the BmNPV genome. Recombinant bacmid-EGFP was obtained through resistance screening and PCR identification. When BmNPV cells reached 40% to 50% of the fullness of a six-well plate, the recombinant bacmid-EGFP was transfected into the cells. The supernatant collected 72 hours after transfection was the EGFP-BmNPV recombinant virus.

[0029] 3. Proliferation Detection of Recombinant Virus EGFP-BmNPV

[0030] (1) Fluorescence microscopy: The recombinant virus EGFP-BmNPV carries the fluorescent reporter gene EGFP. Green fluorescence is observed under a fluorescence microscope, and the density of green fluorescence can reflect the proliferation of the virus.

[0031] (2) Western blot method: Western blot method was used to detect virus proliferation using EGFP monoclonal antibody.

[0032] (3) Virus titer determination: The virus titer was determined by TCID50 method. The specific operation method was as follows: BmN cells were cultured in 96-well plates until the growth density reached about 80%, the culture medium was discarded, and Grace insect culture medium was added in 10-fold serial dilutions (10 -1 ~10 -12100 μL of each recombinant EGFP-BmNPV virus dilution was added, with eight replicates per dilution. After 5 days of incubation at 28°C, cells were observed using an inverted fluorescence microscope. The number of wells infected with each virus dilution that did and did not show virus was recorded. TCID50 was calculated based on the dilution that approximates the 50% infection threshold. Virus titer (TCID50) was calculated using the Karber method: 1g TCID50 = Ld(s - 0.5), where L = the logarithm of the highest dilution, d = the difference between the logarithms of the dilutions, and s = the sum of the positive well ratios.

[0033] 4. Detection of Cellular Autophagy Levels

[0034] Western Blot was used to detect the content of the autophagy marker ATG8-PE, and the ATG8-PE / ATG8 ratio was used to measure the autophagy level of cells.

[0035] 2. Experimental Results

[0036] 1. Drug toxicity to BmN cells

[0037] In order to detect whether the drug is toxic to BmN cells, the CCK8 kit was used to analyze the effect of the drug on cell proliferation. The experimental results showed that drug 1 had no significant effect on cell proliferation at a concentration of 10 μmol / L, inhibited cell proliferation at a concentration of 20 μmol / L, and showed obvious toxicity; drug 2 had no significant effect on cell proliferation at a concentration of 50 μmol / L, and showed obvious cytotoxicity at a concentration of 100 μmol / L; drug 3 had no significant effect on cell proliferation at a concentration of 30 μmol / L, and showed cytotoxicity at a concentration of 50 μmol / L; drug 4 had no cytotoxicity at a concentration of 10 μmol / L, and showed cytotoxicity at a concentration of 20 μmol / L ( Figure 1 ).

[0038] 2. Inhibitory effects of drugs on EGFP-BmNPV proliferation

[0039] The effects of four drugs on BmNPV proliferation were tested at a concentration of 10 μmol / L. The results are as follows: Figure 2 As shown in the figure. After 48 hours of drug treatment, we found that drug 1 and drug 3 significantly inhibited BmNPV proliferation, while drug 2 and drug 4 had no significant inhibitory effect on BmNPV proliferation. We selected drug 1 and drug 3 to further investigate their inhibitory effects on BmN cells.

[0040] 3. Drugs 1 and 3 inhibit autophagy induced by BmNPV infection in BmN cells

[0041] Drugs 1 and 3 have an inhibitory effect on the proliferation of BmNPV. We further verified whether drugs 1 and 3 affect the autophagy level of host cells. The PE-modified bands of ATG8 were detected by Western blot. The results were ( Figure 3 ) found that: a weaker BmATG8-PE band was detected in normally cultured BmN cells, indicating that low-level autophagy occurred in normally cultured BmN cells; after treatment with 5, 10, 20, and 50 μmol / L drug 1 for 24 hours, the BmATG8-PE band weakened with increasing drug concentration, indicating that drug 1 inhibited autophagy in BmN cells, and the inhibition of autophagy increased with increasing concentration in the range of 5-50 μmol / L.

[0042] When EGFP-BmNPV-infected BmN cells were treated with 10 μmol / L of drug 1, and simultaneously infected with virus and treated with drug, the proliferation of virus was observed by fluorescence microscopy at 24h, 36h and 48h. It was found that drug 1 could significantly inhibit the proliferation of virus ( Figure 4 a); Western blot results showed that after drug 1 treatment, the PE modification of BmATG8 protein decreased, indicating that drug 1 inhibited the autophagy level of BmNPV-infected cells ( Figure 4 b, d); TCID at different time points 50 It shows that after 48 hours of drug 1 treatment, the virus titer TCID 50 Significantly lower than DMSO control ( Figure 4 c) in the above example.

[0043] When EGFP-BmNPV-infected BmN cells were treated with 10 μmol / L drug 3, and virus was infected and drug treatment was added at the same time, the proliferation of the virus was observed by fluorescence microscopy at 24h, 36h and 48h. It was found that drug 3 could significantly inhibit the proliferation of the virus ( Figure 5 a); Western blot results showed that drug 3 inhibited BmNPV infection-induced cellular autophagy ( Figure 5 b, c) in the above.

[0044] The above experimental results show that drugs 1 and 3 have antiviral effects on silkworm BmNPV and can be used as new drugs against BmNPV in silkworms.

Claims

1. The use of artesunate in the preparation of a drug for resisting blood-type pus disease in silkworms, characterized in that: The anti-silkworm blood-type pus disease drug is an anti-silkworm nuclear polyhedrosis virus drug.

2. The use according to claim 1, characterized in that The anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cell autophagy.

3. The use of dihydroartemisinin in the preparation of a drug for resisting blood-type purulence of silkworms, characterized in that: The anti-silkworm blood-type pus disease drug is an anti-silkworm nuclear polyhedrosis virus drug.

4. The use according to claim 3, characterized in that The anti-Bombyx mori nuclear polyhedrosis virus drug inhibits the proliferation of Bombyx mori nuclear polyhedrosis virus by inhibiting cell autophagy.

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